PROJECT SUMMARY/ABSTRACT PROJECT 3 Cardiovascular disease, including heart failure, is the most common cause of mortality in adults, and congenital heart defects are the most common form of birth defects in the US. An important concept that has emerged in recent years is that disruptions of cardiac transcription factor networks play important roles in congenital heart defects and in heart failure in adults. MEF2C is one of the core cardiac transcription factors and is required for cardiac development and for postnatal cardiac gene expression and homeostasis. MEF2C functions as signal responsive transcription factor that interacts with numerous co-regulator proteins to control gene expression, yet much remains to be determined about how MEF2C functions in the heart. The most potent transcriptional coactivator for MEF2C described to date is myocardin. MEF2C specifically interacts with a long isoform of myocardin (myocardin-935) to synergistically activate cardiac transcription. Preliminary studies identified a novel bridging mechanism whereby two myocardin-935 molecules interact with MEF2C and with each other via a leucine zipper (LZ) dimerization motif to cooperatively activate paired MEF2 sites, supporting a central role for myocardin dimerization for activation of MEF2-dependent cardiac genes. Furthermore, in silico analyses of cardiac enhancers suggests that paired MEF2 sites are prevalent and occur more frequently than predicted by chance specifically in cardiac enhancers. However, whether these enhancers are bona fide targets of the MEF2C-myocardin complex and how myocardin dimerization influences activity of these enhancers and gene expression in vivo remains to be determined. Additional, unpublished preliminary studies have identified a family with congenital heart defects likely caused by an in-frame microdeletion that results in loss of the myocardin LZ dimerization motif. Similarly, unpublished work shows that mice with an analogous deletion the leucine zipper domain of myocardin die with congenital heart defects. This project will test the hypothesis that the MEF2C-myocardin complex recruits a larger transcriptional coregulatory complex, which is influenced by upstream signaling and myocardin dimerization to regulate cardiac gene expression. To test this overall hypothesis, this project will define the interaction partners of MEF2C and myocardin in the embryonic and adult heart and will identify the phosphorylation sites and other modifications on MEF2C and myocardin. This will provide critical insight into the post-translational regulation of these key cardiac transcription factors. This project will also utilize RNA-seq, ChIP-seq, and other genome- wide approaches from embryos and endogenous tissues to identify transcriptional targets of the MEF2C- myocardin complex and will determine how myocardin dimerization influences the complex and downstream gene expression. Finally, this project will determine the requirement for myocardin dimerization for heart development in vivo by examining the lethal heart development phenotype in myocardin leucine zipper mutant mice and will identify gene expression changes associated with loss of myocardin dimerization.